Method and device for detecting photosynthetic inhibition

ABSTRACT

This invention relates to systems and test strips for detecting the photosynthesis-inhibitory activity of substances by providing cells or cell parts with an intact photosystem, introducing the cells or cell parts into a planar layer, applying the test substance to the planar layer or into the planar layer, excitation of the luminescence of the cells or cell parts in the planar layer by an excitation light source, measuring the luminescence of the cells or the cell parts in the planar layer by means of a detector, and associating the detector signal with the degree of photosynthesis inhibition.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 10/482,990, filed Jan. 7, 2004, which is a national stageapplication of PCT/EP02/07057, filed Jun. 26, 2002, which was publishedin German as International Patent Publication WO 03/006684 on Jan. 23,2003, which was entitled to the right of priority of German PatentApplication 101 33 273.4, filed Jul. 9, 2001.

BACKGROUND OF THE INVENTION

Inhibiting the photosynthesis of plants by herbicidally activesubstances is an important parameter for the ecotoxicological assessmentof substances and also for the search for herbicidally active substancesin crop protection research. Powerful techniques for rapidly measuring aphotosynthesis-inhibitory activity are therefore of great importance inthe ecotoxicological assessment of substances and as screening methodsfor the search for novel crop protectants.

A variety of tests, both on higher plants and on microalgae, are knownfor assaying photosynthesis-inhibitory activity. The known measuringprinciples are based, inter alia, on the fluorescence of chlorophyll oron measuring the photosynthesis oxygen production (B. Hock, C. Fedtke,R. R. Schmidt, Herbizide [Herbicides], Georg Thieme Verlag, 1995, 54 and112-114; D. Merz, M. Geyer, D. A. Moss, H.-J. Ache, Fresenius J. Anal.Chem, 1996, 354: 299-305). All of these methods, which represent theprior art, have limitations which do not permit high-throughputmeasurements as they are carried out in the screening of activeingredients, miniaturization, for example a high degree ofparallerlization, or a direct couplings with analytical separationtechniques for detecting activities in substance mixtures.

Measuring the fluorescence of chlorophyll is an established standardmethod for studying the photosynthesis process. The methods used in thiscontext rely on fluorimeters which, owing to their methodology, which isbased on measurements using probes or cuvettes, only permit serialmeasurements and are therefore not suitable for high-throughputapplications. Moreover, such methods are also very difficult tominiaturize. Typical instruments for this technique are available fromthe manufacturers mentioned hereinbelow, among others: ADC BioScientificLtd., Hansatech Instruments, Heinz Walz GmBH, Qubit Systems Inc.

In the DF-Algentest [DF algae test], water samples are treated withgreen algae and subsequently measured luminometrically (Methoden derbiologischen Wasseruntersuchung [Methods of Biological Water Analytics],Volume 2: Biologische Gewässeruntersuchung, G. Fischer Verlag, 1999,page 386-388). In this test, the first step is the determination ofdeactivation kinetics for the lagging luminescence of the photosynthesispigment complex. Conclusions regarding the presence ofphotosynthesis-inhibitory substances are drawn by comparison with thecorresponding deactivation kinetics for an untreated reference sample.This method is only capable of processing samples in series and is thusnot suitable for high-throughput measurements.

A further limitation relates to the sample volume for the DF-Algentest,which, owing to the dimensions of the equipment, is in the milliliterorder. This method does not allow miniaturization. Moreover, substancemixtures, as are typical for realistic samples, can only be assessed intheir entirety by this method. Owing to the possibility of interactionsbetween the sample constituents, there is a risk of false positives.

Tests on higher plants are also known (see, for example, W. Bilger, U.Schreiber, M. Bock, Oecologia 102, 1995, pp. 425-432). These testsprovide findings on photosynthetic inhibition via a method in which thefluorescence is measured. Again, the geometry of the test deviceprevents a high degree of parallelization and miniaturization. Again,substance mixtures can only be assessed in their entirety.

EP 588 139 A1 describes a test for substance mixtures. The biologicaleffect of the substances in a substance mixture is tested by acombination of chromatographic separation of the substance mixture intothe substances to be tested in chromatographic zones, followed by abioassay (toxicity) of the individual fractions which have beenseparated. In the bioassay, the individual fractions are brought intocontact with luminescence microorganisms which indicate the biologicaleffect of this fraction by means of a local change in theirbioluminescence at the individual fractions.

The possibility of parallelization and miniaturization of activityassays is described in EP 1 043 582 A2. According to the methoddisclosed in EP 1 043 582 A2, a sensor layer consisting of adiffusion-controlling matrix in which activity sensors are suspended isemployed. The bioactivity of the test substances is indicated by opticalsignals upon contact of this sensor layer with samples.

The object of the invention consists in providing a device and a methodfor detecting photosynthesis-inhibitory substances which makes possibleminiaturization and a markedly higher sample throughput in comparisonwith the prior art.

The object of the invention is achieved by a method for detecting thephotosynthesis-inhibitory activity of substances, comprising thefollowing steps:

-   providing cells or cell parts with an intact photosystem,-   introducing the cells or cell parts into a planar layer,-   applying the test substance to the planar layer or into the planar    layer,-   exciting the luminescence of the cells or cell parts in the planar    layer by an excitation light source,-   measuring the luminescence of the cells or cell parts in the planar    layer by means of a detector, and-   associating the detector signal with the degree of photosynthesis    inhibition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a fluorescent image obtained according to Example 1.

FIG. 2 illustrates a fluorescent image obtained according to Example 2.

DETAILED DESCRPTION OF THE INVENTION

The cells may be derived from algae, microalgae, bacteria, in particularcyanobacteria, which has a photosynthesis system, plant cell cultures orplant homogenate. The method also operates with cells whose vitality isdamaged, as long as an intact photosystem II (PS II) is present.

The cells may also originate from selected mutants or from geneticallymodified organisms.

The planar layer is preferably a gel layer. The planar layer preferablyhas a thickness in the range of from 0.1 mm to 10 mm. The cells or cellparts can be introduced into a planar layer by embedding for examplegreen algae in agarose or acrylate gels or other gellants or viscousmedia.

Application of the test substance to the planar layer or into the planarlayer is effected for example by syringe techniques or pin tools orsuitable pressure techniques (jet systems and the like), preferably inthe form of spots.

The luminescence is fluorescence and/or phosphorescence (laggedluminescence). Measuring the phosphorescence is advantageous overmeasuring the fluorescence since there is no need to discriminatebetween excitation and emission. On the other hand, measurement withfluorescence is advantageous owing to its higher sensitivity.

Suitable excitation light sources are not only white-light sources, forexample halogen light or fluorescent tubes, but also light sources whichemit within a narrow spectral range, for example light-emitting diodes.Daylight may also act as the excitation light source. Excitation may becontinuous or in a pulsed mode (pulse modulation technique).

Detection is effected with instruments which are capable of imaging theemitted luminescence in a wavelength range of >680 nm with sufficientsensitivity (for example Vidicon System, CCD camera, scanner,phosphorimager, photographic film).

Time-resolved light measurements, if appropriate together with pulsedexcitation and correlation of excitation and measurement, may also becarried out.

Independently of excitation exposure, supplementary illumination or adark phase for controlling the photosynthesis activity may be employed.

Photosynthesis-inhibitory test substances which are applied to or ontothe planar layer according to the invention affect the luminescentbehavior of the photosynthesis pigment complex. Spots of photosynthesisinhibitors, such as atrazine, which are applied to the planar layer canbe detected simply, and in the case of a large number of spots,simultaneously via their activity, for example by a significantweakening of the lagged luminescence (phosphorescence), usingvideoimaging methods. As an alternative, the increased fluorescence ofthe photopigments when the photosystem II is inhibited may also be usedfor imaging the PS II-active substance spots.

The invention furthermore relates to a system for detecting thephotosynthesis-inhibitory activity of substances by the method accordingto the invention, comprising

-   a planar layer with cells or cell parts with an intact photosystem,-   means for applying the test substance to the planar layer or into    the planar layer,-   excitation light source for exciting the luminescence of the cells    or the cell parts in the planar layer,-   detector for measuring the luminescence of the cells or cell parts    in the planar layer,-   evaluation means for associating the detector signal with the degree    of photosynthesis inhibition.

The means for applying the test substance to the planar layer or intothe planar layer can be, for example syringe systems, steel needles (pintools) or suitable pressure stamps, and also jet systems.

The evaluation can be effected visually or by means of suitable imagingtechniques.

The invention furthermore relates to a test strip or sensor chip fordetecting the phososynthesis-inhibitory activity of substances by themethod according to the invention comprising a planar layer with cellsor cell parts with an intact photosystem, where, after the testsubstance has been applied to the planar layer or into the planar layer,and after the subsequent excitation of the luminescence of the cells orthe cell parts in the planar layer by an excitation light source, andafter measuring the luminescence of the cells or the cell parts in theplanar layer with a detector, the degree of photosynthesis inhibitioncan be determined on the basis of the detector signal.

The planar layer of the test strip or sensor chip consists preferably ofgreen algae in agarose or acrylate gels. In this manner, stabledetection layers can be prepared which retain their function as testsystem for a photosynthesis-inhibitory activity even when stored overprolonged periods.

An advantage of the method according to the invention is the high degreeof miniaturization and parallelization of the detection method forphotosynthesis-inhibitory substances. Parallelization allows a highsample throughput to be achieved. Miniaturization allows one to make dowith considerably less material.

The method according to the invention makes it possible to apply severalthousands of substance spots to an area of 9 cm*12 cm and thus toachieve not only parallelization, but a degree of miniaturization of <10ng of test substance in less than 500 nl of test volume.

The space-resolving analysis permits the identification ofphotosynthesis-inhibitory substances as components of substance mixturesin thin-layer chromatograms or electropherograms in a trouble-freemanner by first subjecting the substance mixture to chromatographic orelectrophoretic separation on a thin-layer chromatography plate or anelectrophoresis layer and subsequently studying thephotosynthesis-inhibitory activity of the fractions by the methodaccording to the invention.

The space-resolving analysis also permits the application ofphotosynthesis-inhibitory substances to different positions of a supportand to study the photosynthesis-inhibitory activity of these spots bythe method according to the invention.

The method according to the invention can be employed in activeingredient research for the optimization of photosynthesis inhibitors. Afurther field of application is, for example, the specific measurementof the herbicidal activity in wastewater and environmental samples whichcan be attributed to pollutants.

EXAMPLES Example 1

Example 1 shows the specific detection by the method according to theinvention of photosynthesis-inhibitory substances by means of inducedfluorescence in the layer system according to the invention.

A thin agarose layer (layer thickness approx. 4 mm) in which green algae(Scenedesmus subspicatus) had been suspended, was employed to detect thephotosynthesis-inhibitory action.

The green algae were grown as follows:

The algae from a Scenedesmus subspicatus stock are used to inoculate 50ml of growth medium in a sterile 100 ml Erlenmeyer flask. The solutionis subsequently incubated for 7 days at 23° C. and 125 rpm in acontrolled-environment cabinet equipped with fluorescent tubes, withexposure to light.

The growth medium contains:

-   58 mg/l sodium carbonate-   496 mg/l sodium nitrate-   39 mg/l potassium hydrogenphosphate-   75 mg/l magnesium sulfate heptahydrate-   36 mg/l calcium chloride dihydrate-   10 mg/l Titriplex III-   3 mg/l citric acid    and is brought to pH 7.5±0.2 using 1 N HCl and/or 1 N NaOH. Prior to    use, the medium is autoclaved for 20 minutes at 121° C.

Preparation of the algal layer:

25 ml of the algal suspension (optical density approx. 2 mAU) are mixedat temperatures of below 40° C. with 15 ml of 1% strength agarose MPsolution (Boehringer Mannheim GmbH Art. No. 1388983) until homogeneous.Before this suspension is cold, it is placed into a single-well plate(Nalge Nunc, Omni Tray Single Well 86×128 mm), where a gel layer withuniformly suspended algae is formed upon cooling. This detection layercan be employed immediately or else after several weeks' storage formeasuring the photosynthesis-inhibitory action.

Substance transfer to the detection layer, and incubation:

To carry out the parallel activity test according to the invention, thealgal layer is stamped with test substances from a microtiter plate,using a 96-fold pin tool (Nalge Nunc 96 Pin Replicator). The sampledeposit on the microtiter plate (see Table 1) thus also lays down theposition of the substance in question on the algal layer. The testsubstances were present in the microtiter plate in the form of a DMSOsolution (100 μmol substance in 100 μl per well). Using the pin tool, ineach case approx. 0.5 μl of sample solution were transferred to thedetection layer per pin. Prior to the fluorescence measurement thestamped detection layer was incubated for 15 minutes at roomtemperature.

TABLE 1 Parallel detection of the activity of substances by fluorescenceimaging on a 96-well microtiter plate with algal layer PositionSubstance Activity A1 B1 Glyphosate C1 Thidiazuron D1 Pendimethalin E1Fluazifop-P-butyl F1 Thifensulfuron-methyl G1 Quinmerac H1 Ioxynil A2MCPA B2 Tebuthiuron X C2 Diuron X D2 Mefenacet E2 Cyanazine X F2Oxadiazon G2 Terbuthylazine X H2 Diflufenican A3 Dicamba B3 AcifluorfenC3 Ametryne X D3 Prometon X E3 Prometryne X F3 Sulfometuron-methyl G3 —H3 Metribuzin X A4 Pyrazolate B4 Norflurazon C4 Linuron X D4 EPTC E4Metazachlor F4 Metamitron X G4 Naproamid H4 Bentazone A5 Pyridate B5 —C5 Pretilachlor D5 Sethoxydim E5 Isoproturon X F5 Nicosulfuron G5Bromacil X H5 Haloxyfop-P-methyl A6 Phenmedipham X B6 Alachlor C6 — D6Thiobencarb E6 Difenzoquat F6 Imazapyr G6 Metsulfuron-methyl H6Metolachlor A7 Propanil X B7 Clopyralid C7 Bensulfuron-methyl D7 — E7Atrazine X F7 Simazine X G7 — H7 Propyzamid A8 Quinchlorac B8 Diquat C8Bifenox D8 Glufosinate E8 Butylate F8 Ethalfluralin G8 Sulcotrione H8Tralkoxydim A9 Amitrole B9 — C9 Butachlor D9 Hexazinon X E9 Alloxydim F9Chlorimuron-ethyl G9 — H9 Mecoprop A10 Fluometuron X B10Fenoxaprop-P-ethyl C10 Desmedipham X D10 Primisulfuron E10 Di-allate F10Asulam G10 — H10 Ethofumesate F12 50 ng Metamitron Reference for PSIIinhibition G12 125 ng Metamitron Reference for PSII inhibition H12 250ng Metamitron Reference for PSII inhibition

Parallel detection of the activity by fluorescent imaging:

A videoimaging system (Molecular Light Imager NightOWL by PerkinElmerLife Sciences) was employed for recording the fluorescence image. Tocarry out the measurement, the single-well plate was placed on a lighttable whose white-light source was limited to wavelengths of below 475nm using a filter (Omega 475 RDF 40). For the selective detection of thefluorescence light, the camera lens was equipped with a filter whichallows light above 680 nm to pass through (Andover P/N: 680FS10-50).Fluorescence excitation and recording by the camera were carried outsimultaneously over a period of 1 second. The fluorescent image wasevaluated visually on the screen of the videoimaging system. Fordocumentation purposes, TIFF files were formatted with suitable graphicsprograms and labeled (Adobe Photoshop 5.0, MS Powerpoint 97).

Results:

The fluorescent image revealed 22 light spots (see FIG. 1). Thesubstances deposited at these spots bring about an increasedfluorescence owing to their interaction with the photosystem.Metamitron, a known photosynthesis inhibitor, has been applied asreference substance to positions F12, G12 and H12 in amounts of 50 ng,125 ng and 250 ng. All of the substances which were noted in thisparallel assay and the substances marked X in Table 1 are knowninhibitors of the photosystem II.

Example 2

Example 2 shows the specific detection by the method according to theinvention of photosynthesis-inhibitory substances by means ofphosphorescence in the layer system according to the invention.

The algal layer was prepared analogously to Example 1.

The same test substances as in Example were applied to the algal layerin identical positions. Incubation was likewise for 15 minutes.

Parallel detection of the activity by phosphorescent imaging:

A videoimaging system (Molecular Light Imager NightOWL by PerkinElmerLife Sciences) was employed for recording the phosphorescence image. Forthe measurement, the NUNC plate was placed on a light table which wasequipped with a white light source. To record the phosphorescent light,no filters were inserted before the camera lens. The algal layer wasexposed for 90 seconds in order to excite phosphorescence. After aperiod of 15 seconds, the image was taken by the camera with an exposuretime of 30 seconds. The phosphorescent image was evaluated visually onthe screen of the videoimaging system. For documentation purposes, TIFFfiles were formatted with suitable graphics programs and labeled (AdobePhotoshop 5.0, MS Powerpoint 97).

Results:

The phosphorescent image revealed 22 dark spots (see FIG. 2). Thesubstances deposited at these spots bring about more rapid deactivationof the phosphorescence owing to their interaction with the photosystem.Metamitron, a known photosynthesis inhibitor, has been applied asreference substance to positions F12, G12 and H12 in amounts of 50 ng,125 ng and 250 ng. The results are in good agreement with the results ofthe fluorescent imaging (see also Example 1). All of the substanceswhich were noted in this parallel assay are known photosystem IIinhibitors.

1-16. (canceled)
 17. A system for detecting thephotosynthesis-inhibitory activity of substances, comprising a planarlayer with cells or cell parts with an intact photosystem, means forapplying the test substance to the planar layer or into the planarlayer, an excitation light source for exciting the luminescence of thecells or the cell parts in the planar layer, a detector for measuringthe luminescence of the cells or cell parts in the planar layer,evaluation means for associating the detector signal with the degree ofphotosynthetic inhibition.
 18. A system as claimed in claim 17, whereinthe means for applying the test substance to the planar layer or intothe planar layer or to the support of the planar layer is a syringesystem, a pin tool or a suitable pressure stamp or a jet system.
 19. Asystem as claimed in claim 17, wherein the detector employed is, forexample, a Vidicon system, a CCD camera, a scanner, a phosphorimager ora photographic film.
 20. A system as claimed in 17, wherein theevaluation is carried out by imaging or visually.
 21. A system asclaimed in 17, wherein support for the planar layer is a thin-layerchromatography plate or an electrophoresis layer with substance zonesdeposited thereon.
 22. A test strip or sensor chip for detecting thephotosynthesis-inhibitory activity of substances comprising a planarlayer with cells or cell parts of an intact photosystem, with which,after application of the test substance to the test strip or sensorchip, after subsequent excitation of the luminescence of the cells orthe cell parts in the planar layer by an excitation light source, andafter measuring the luminescence of the cells or cell parts in theplanar layer by means of a detector, the degree of photosynthesisinhibition of the test substance can be determined on the basis of thedetector signal.
 23. A test strip or sensor chip as claimed in claim 22,wherein the planar layer of the test strip or sensor chip consists ofgreen algae in agarose or acrylate gel or in a viscous solution.
 24. Atest strip or sensor chip as claimed in claim 22, wherein the cells areavital cells in which the photosynthesis system II remains intact.